Solubilized eggshell membrane supplies a type III collagen-rich elastic dermal papilla.

Signs of aging in facial skin correlate with lifespan and chronic disease; however, the health of aging skin has not been extensively studied. In healthy young skin, the dermis forms a type III collagen-rich dermal papilla, where capillary vessels supply oxygen and nutrients to basal epidermal cells. Chicken eggshell membranes (ESMs) have been used as traditional medicines to promote skin wound healing in Asian countries for many years. Previously, we designed an experimental system in which human dermal fibroblasts (HDFs) were cultured on a dish with a solubilized ESM (S-ESM) bound to an artificial phosphorylcholine polymer; we found that genes that promoted the health of the papillary dermis, such as those encoding type III collagen, were induced in the S-ESM environment. The present study found that a gel with a ratio of 20% type III/80% type I collagen, similar to that of the baby skin, resulted in a higher elasticity than 100% type I collagen (p < 0.05) and that HDFs in the gel showed high mitochondrial activity. Thus, we decided to perform further evaluations to identify the effects of S-ESM on gene expression in the skin of hairless mice and found a significant increase of type III collagen in S-ESM. Picrosirius Red staining showed that type III collagen significantly increased in the papillary dermis after S-ESM treatment. Moreover, S-ESM application significantly improved human arm elasticity and reduced facial wrinkles. ESMs may have applications in extending lifespan by reducing the loss of tissue elasticity through the increase of type III collagen.

Hydrolyzed eggshell membrane immobilized on phosphorylcholine polymer supplies extracellular matrix environment for human dermal fibroblasts.

We have found that a water-soluble alkaline-digested form of eggshell membrane (ASESM) can provide an extracellular matrix (ECM) environment for human dermal fibroblast cells (HDF) in vitro. Avian eggshell membrane (ESM) has a fibrous-meshwork structure and has long been utilized as a Chinese medicine for recovery from burn injuries and wounds in Asian countries. Therefore, ESM is expected to provide an excellent natural material for biomedical use. However, such applications have been hampered by the insolubility of ESM proteins. We have used a recently developed artificial cell membrane biointerface, 2-methacryloyloxyethyl phosphorylcholine polymer (PMBN) to immobilize ASESM proteins. The surface shows a fibrous structure under the atomic force microscope, and adhesion of HDF to ASESM is ASESM-dose-dependent. Quantitative mRNA analysis has revealed that the expression of type III collagen, matrix metalloproteinase-2, and decorin mRNAs is more than two-fold higher when HDF come into contact with a lower dose ASESM proteins immobilized on PMBN surface. A particle-exclusion assay with fixed erythrocytes has visualized secreted water-binding molecules around the cells. Thus, HDF seems to possess an ECM environment on the newly designed PMBN-ASESM surface, and future applications of the ASESM-PMBN system for biomedical use should be of great interest.

Dynamic localization of αB-crystallin at the microtubule cytoskeleton network in beating heart cells.

αB-crystallin is highly expressed in the heart and slow skeletal muscle; however, the roles of αB-crystallin in the muscle are obscure. Previously, we showed that αB-crystallin localizes at the sarcomere Z-bands, corresponding to the focal adhesions of cultured cells. In myoblast cells, αB-crystallin completely colocalizes with microtubules and maintains cell shape and adhesion. In this study, we show that in beating cardiomyocytes α-tubulin and αB-crystallin colocalize at the I- and Z-bands of the myocardium, where it may function as a molecular chaperone for tubulin/microtubules. Fluorescence recovery after photobleaching (FRAP) analysis revealed that the striated patterns of GFP-αB-crystallin fluorescence recovered quickly at 37°C. FRAP mobility assay also showed αB-crystallin to be associated with nocodazole-treated free tubulin dimers but not with taxol-treated microtubules. The interaction of αB-crystallin and free tubulin was further confirmed by immunoprecipitation and microtubule sedimentation assay in the presence of 1-100 µM calcium, which destabilizes microtubules. Förster resonance energy transfer analysis showed that αB-crystallin and tubulin were at 1-10 nm apart from each other in the presence of colchicine. These results suggested that αB-crystallin may play an essential role in microtubule dynamics by maintaining free tubulin in striated muscles, such as the soleus or cardiac muscles.

Analysis of the alphaB-crystallin domain responsible for inhibiting tubulin aggregation.

The cytoskeleton has a unique property such that changes of conformation result in polymerization into a filamentous form. alphaB-Crystallin, a small heat shock protein (sHsp), has chaperone activities for various substrates, including proteins constituting the cytoskeleton, such as actin; intermediate filament; and tubulin. However, it is not clear whether the "alpha-crystallin domain" common to sHsps also has chaperone activity for the protein cytoskeleton. To investigate the possibility that the C-terminal alpha-crystallin domain of alpha-crystallin has the aggregation-preventing ability for tubulin, we constructed an N-terminal domain deletion mutant of alphaB-crystallin. We characterized its structural properties and chaperone activities. Far-ultraviolet (UV) circular dichroism measurements showed that secondary structure in the alpha-crystallin domain of the deletion mutant is maintained. Ultracentrifuge analysis of molecular masses indicated that the deletion mutant formed smaller oligomers than did the full-length protein. Chaperone activity assays demonstrated that the N-terminal domain deletion mutant suppressed heat-induced aggregation of tubulin well. Comparison of chaperone activities for 2 other substrates (citrate synthase and alcohol dehydrogenase) showed that it was less effective in the suppression of their aggregation. These results show that alphaB-crystallin recognizes a variety of substrates and especially that alpha-crystallin domain binds free cytoskeletal proteins. We suggest that this feature would be advantageous in its functional role of holding or folding multiple proteins denatured simultaneously under stress conditions.